1. Field of the Invention
The present invention relates to a novel DNA fragment carrying a toluene monooxygenase gene, a novel recombinant DNA containing the DNA fragment, a transformant containing the recombinant DNA, and a method for degrading chlorinated aliphatic hydrocarbon compounds such as trichloroethylene (TCE) and dichloroethylene (DCE) and aromatic compounds such as toluene, benzene, phenol, and cresol. The present invention also relates to a method for environmental remediation useful for cleaning of aqueous media such as wastewater and effluent containing at least either a chlorinated aliphatic hydrocarbon compound or an aromatic compound and air (gas phase) and soil polluted with chlorinated aliphatic hydrocarbon compounds.
2. Related Background Art
Recently, it has become a serious problem the environmental pollution with volatile organic chlorinated compounds which are harmful to the organisms and hardly degradable. Especially, the soil in the industrial areas in Japan as well as abroad is considered to be contaminated with chlorinated aliphatic hydrocarbon compounds such as tetrachloroethylene (PCE), trichloroethylene (TCE), and dichloroethylene (DCE) and aromatic compounds such as toluene, benzene, phenol, and cresol. In fact, there have been a number of reports on actual detection of such pollutants through environmental surveys. It is supposed that these compounds remaining in soil dissolve in ground water via rainwater, and thereby spread over the surrounding areas. There is a strong suspicion that these compounds are carcinogens, and further, these are quite stable in the environment; therefore contamination of groundwater, which is used as a source of drinking water, has become a serious social problem. Therefore, cleaning of soil and aqueous media such as contaminated groundwater by removal and degradation of these compounds and accompanying cleaning of the surrounding gas phase is quite important in view of the environment protection, and technologies for remedying the environment (for example, adsorption treatment using activated carbon, degradation treatment using light and heat) have been developed. Current technologies, however, are not always practical in terms of cost and operability. Recently, microbial degradation of chlorinated aliphatic hydrocarbon compounds such as TCE that is stable in environment has been reported. The microbial degradation method has advantages such as: (1) degradation of chlorinated aliphatic hydrocarbon compounds into harmless substances by using appropriately selected microorganism; (2) no requirement for any special chemicals in principle; and (3) reduction of the labor and costs of maintenance.
The examples of microorganisms capable of degrading TCE are as follows:
Welchia alkenophila sero 5 (U.S. Pat. No. 4,877,736, ATCC 53570, Welchia alkenophila sero 33 (U.S. Pat. No. 4,877,736, ATCC 53571), Methylocystis sp. Strain M (Agric. Biol. Chem., 53, 2903 (1989), Biosci. Biotech. Bichem., 56, 486 (1992), ibid. 56, 736 (1992)), Methylosinus trichosporium OB3b (Am. Chem. Soc. Natl. meet. Div. Environ. Microbiol., 29, 365 (1989), Appl. Environ. Microbiol., 55, 3155 (1989), Appl. Biochem. Biotechnol. 28, 877 (1991), Japanese Patent Application Laid-Open No. 2-92274 specification, Japanese Patent Laid-Open Application No. 3-292970), Methylomonas sp. MM2 (Appl. Environ. Microbiol., 57, 236 (1991), Alcaligenes denitrificans ssp. Xylosoxidans JE75 (Arch. Microbiol., 154, 410 (1990), Alcaligenes eutrophus JMP134 (Appl. Environ. Microbiol., 56, 1179 (1990), Alcaligenes eutrophus FERM-13761 (Japanese Patent Laid-Open Application No. 7-123976), Pseudomonas aeruginosa J1104 (Japanese Patent Application Laid-Open No. 7-236895), Mycobacterium vaccae JOB5 (J. Gen. Microbiol., 82, 163 (1974), Appl. Environ. Microbiol., 55, 2960 (1989), ATCC 29678), Pseudomonas putida BH (Gesuidou Kyoukai-shi (Japan Sewage Works Association Journal), 24, 27 (1987)), Pseudomonas sp. strain G4 (Appl. Environ. Microbiol., 52, 383 (1968), ibid. 53, 949 (1987), ibid. 54, 951 (1988), ibid. 56, 279 (1990), ibid. 57, 193 (1991), U.S. Pat. No. 4,925,802, ATCC 53617, this strain was first classified as Pseudomonas cepacia and then changed to Pseudomonas sp.), Pseudomonas mendocia KR-1 (Bio/Technol., 7, 282 (1989)), Pseudomonas putida F1 (Appl. Environ Microbiol., 54, 1703 (1988), ibid. 54, 2578 (1988)), fluorescens PFL12 (Appl. Environ. Microbiol., 54, 2578 (1988)), Pseudomonas putida KWI-9 (Japanese Patent Application Laid-Open No. 6-70753), Burkholderia cepacia KK01 (Japanese Patent Application Laid-Open No. 6-22769), Nitrosomonas europaea (Appl. Environ. Microbio., 56, 1169 (1990), Lactobacillus vaginalis sp. nov (Int. J. Syst. Bacteriol., 39, 368 (1989), ATCC 49540), Nocardia corallina B-276 (Japanese Patent Application Laid-Open No. 8-70881, FERM BP-5124, ATCC 31338), and so on.
The problem in actually using these degrading microorganisms in environmental remediation treatment, however, resides in optimizing and maintaining expression of their degradation activity for chlorinated aliphatic hydrocarbon compounds such as TCE. In an environmental remediation treatment which utilizes phenol, toluene, methane, or the like as an inducer, continuous supply of the inducer is indispensable, since depletion of such inducers directly results in stoppage of degradation of chlorinated aliphatic hydrocarbon compounds. Presence of such inducers, on the other hand, may inhibit the efficient degradation of the target substance such as TCE, since the affinity of the chlorinated aliphatic hydrocarbon compounds such as TCE as a substrate is considerably low in comparison with these inducers. In addition, precise control of the inducer concentration on the treatment spot is difficult.
Thus, use of an inducer is a large problem in practical application of environmental remediation treatment utilizing microorganisms.
In order to solve the problem, Nelson et al. developed a method using tryptophan as an inducer for degradation of volatile organic chlorinated compounds (Japanese Patent Application Laid-Open No. 4-502277). Tryptophan, however, is a very expensive substance, and although tryptophane has no toxicity or risk as a substance, it is not preferable to introduce excessive carbon and nitrogen sources into environment since it may induce eutrophication. In addition, the problem that tryptophan serves as a competitive inhibitor in degradation of TCE still remains.
Shields et al. obtained a mutant strain of Pseudomonas cepacia G4 (changed to Pseudomonas sp. upon deposition to ATCC) by the transposon technique, which mutant strain does not require an inducer (in this case, phenol or toluene) and can degrade TCE (Appl. Environ. Microbiol., 58, 3977 (1992), International Publication No. WO/19738). Also, a mutant not requiring methane as the inducer has been isolated from Methylosinus trichosporium OB3b, a methanotroph capable of degrading TCE (U.S. Pat. No. 5,316,940).
Japanese Patent Application Laid-Open No. 8-294387 also discloses strain JM1 (FERM BP-5352) capable of degrading volatile organic chlorinated compounds and aromatic compounds without requiring an inducer, isolated by nitrosoguanidine mutagenization of strain J1 (FERM BP-5102). While, it has been studied to introduce resting cells expressing TCE-degrading activity into the remediation site after the preculture of the cells in the presence of an inducer (Environ. Sci. Technol., 30, 1982 (1996)).
It has been reported that remediation treatment not requiring the inducer actually makes the remediation treatment easy and efficient compared to the conventional treatment using inducers.
However, the growth control of the degrading microorganisms is very important for both the expression of the degradation activity on demand and the continuation of degradation. When resting cells are used, it is a problem to be solved that TCE cannot be degraded beyond the amount and period of degradation capacity of the introduced resting cells. In addition, in a large scale treatment, there are further problems that degradation activity will decrease since it takes a long time to prepare resting cells; the treating apparatus must be large in scale; treatment process is complicated; and the cost may be unfavorably high. Accordingly, it has been attempted to introduce a plasmid carrying a DNA fragment containing a gene region encoding oxygenase or hydroxylase into a host microorganism to make the host express the TCE degradation activity constitutively or inducibly using a harmless inducer. For example, there are Pseudomonas mendocina KR-1 (Japanese Patent Application Laid-Open No. 2-503866, Pseudomonas putida KWI-9 (Japanese Patent Application Laid-Open No. 6-105691), Pseudomonas putida BH (Summary of 3rd Conference on Pollution of Ground Water/Soil and Its Protective Countermeasure, p.213 (1994)), and a transformant carrying both a toluene degradation enzyme gene derived from Pseudomonas putida F1 and a biphenyl degradation enzyme gene derived from Pseudomonas pseudoalkaligenes (Japanese Patent Application Laid-Open No. 7-143882). However, the reported TCE degradation activity of the transformants are low, and the advantages of the transformants has not been fully exploited for efficient degradation of TCE, such as the ease of degradation control, freedom in designing recombinant, and no requirements for inducers, far from efficient TCE degradation.
It is an object of the present invention to provide a novel DNA fragment encoding a toluene monooxygenase of a high efficiency in degrading aromatic compounds and/or organic chorine compounds, a novel recombinant DNA containing the DNA fragment, and a transformant containing the recombinant DNA. It is another object of the present invention to provide an efficient biodegradation method for halogenated aliphatic compounds such as trichloroethylene (TCE) and dichloroethylene (DCE) and aromatic compounds such as toluene, benzene, phenol, and cresol using the transformant, specifically an efficient environmental remediation method useful for cleaning aqueous media such as wastewater and effluent containing halogenated aliphatic hydrocarbon compounds and/or aromatic compounds, remedying soil polluted with halogenated aliphatic hydrocarbon compounds or aromatic compounds, and cleaning air (gas phase) polluted with halogenated aliphatic hydrocarbon compounds.
The inventors of the present invention isolated a microorganism Ralstonia eutropha strain TB64 having a toluene monooxygenase that oxidizes toluene to ortho-cresol and 3-methylcatechol and deposited it in the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology in accordance with the requirements of the Budapest Treaty, Deposit Date: Sep. 3, 1998, Accession No. FERM BP-6933). The inventors strained to isolate the toluene monooxygenase gene from the above strain TB64 and successful isolation and characterization of the gene completed the present invention.
According to one aspect of the present invention, there is provided a DNA fragment of about 5.3 Kb containing a toluene monooxygenase gene, having 3 BamHI, 1 ClaI, 1 EcoRI, 3 KpnI, 2 NcoI, 2 NspV, 2 ScaI, 2 SmaI, 2 SphI, 1 StuI, 0 DraI, 0 EcoRV, 0 HindIII, 0 HpaI, 0 NdeI, 0 PvuII, o ScaI, 0 Sse83871, 0 XbaI, 0 XhoI restriction sites, and having a restriction map of: 
According to another aspect of the present invention, there is provided a DNA fragment having a nucleotide sequence of SEQ ID NO: 1 in the Sequence Listing.
According to another aspect of the present invention, there is provided a DNA fragment having a nucleotide sequence of SEQ ID NO: 1 with deletion, substitution, and/or addition of one or more nucleotides encoding a protein having a toluene monooxygenase activity.
According to another aspect of the present invention, there is provided a recombinant DNA comprising a vector which can be replicate or maintained in a host and the above mentioned DNA fragment.
According to another aspect of the present invention, there is provided a DNA fragment containing a portion encoding a toluene monooxygenase, the portion comprising a region encoding a polypeptide TomL having an amino acid sequence of SEQ ID NO: 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO: 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO: 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO: 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO: 7 of the Sequence Listing, and the regions are aligned so that expressed TomL-TomP polypeptides can form an active monooxygenase protein.
According to still another aspect of the present invention, there is provided a recombinant DNA comprising a vector, a promoter, and the above mentioned DNA fragment, wherein the vector and the promoter are functionally ligated to the DNA fragment to enable expression of the toluene monooxygenase encoded by the DNA fragment in the host.
According to still another aspect of the present invention, there is provided a DNA fragment comprising a region encoding a polypeptide TomK which has an amino acid sequence of SEQ ID NO: 2 and a property to enhance the toluene monooxygenase activity of a protein comprised of at least TomL-TomP; or a region encoding a variant TomK in which the amino acid sequence of SEQ ID NO: 2 is altered with the proviso that the property to enhance the toluene monooxygenase activity is not impaired.
According to still another aspect of the present invention, there is provided another recombinant DNA comprising a vector, a promoter, a first DNA fragment and a second DNA fragment;
wherein the first DNA encodes a toluene monooxygenase and comprises a region encoding a polypeptide TomL having an amino acid sequence of SEQ ID NO: 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO: 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO: 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO: 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO: 7 of the Sequence Listing,
the second DNA fragment encodes a polypeptide TomK having an amino acid sequence of SEQ ID NO: 2 and a property to enhance the toluene monooxygenase activity of a protein comprised of at least TomL to TomP; or encodes a variant TomK polypeptide in which the amino acid sequence of SEQ ID NO: 2 is altered with the proviso that the property to enhance the toluene monooxygenase activity is not impaired;
the first DNA fragment is functionally linked to the promoter to express the toluene monooxygenase activity, and the second DNA fragment is functionally linked to the promoter to express the property to enhance the activity of the toluene monooxygenase encoded by the first DNA fragment.
According to still another aspect of the present invention, there is provided a recombinant DNA comprising a vector; a first promoter and a first DNA fragment; and a second promoter and a second DNA fragment;
wherein the first DNA encodes a toluene monooxygenase and comprises a region encoding a polypeptide TomL having an amino acid sequence of SEQ ID NO: 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO: 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO: 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO: 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO: 7 of the Sequence Listing,
the second DNA fragment encodes a polypeptide TomK having an amino acid sequence of SEQ ID NO: 2 and a property to enhance the toluene monooxygenase activity of a protein comprised of at least TomL to TomP; or encodes a variant TomK polypeptide in which the amino acid sequence of SEQ ID NO: 2 is altered with the proviso that the property to enhance the toluene monooxygenase activity is not impaired;
the first DNA fragment is functionally linked to the first promoter to express the toluene monooxygenase activity, and the second DNA fragment is functionally linked to the second promoter to express the property to enhance the activity of the toluene monooxygenase encoded by the first DNA fragment.
According to still another aspect of the present invention, there is provided a transformant obtainable by introducing a recombinant DNA into a host microorganism, the recombinant DNA comprising a vector which can replicate or maintained in the host and ligated to a DNA fragment of about 5.3 Kb containing a toluene monooxygenase gene, having 3 BamHI, 1 ClaI, 1 EcoRI, 3 KpnI, 2 NcoI, 2 NspV, 2 ScaI, 2 SmaI, 2 SphI, 1 StuI, 0 DraI, 0 EcoRV, 0 HindIII, OHpaI, 0 NdeI, 0 PvuII, 0 ScaI, 0 Sse83871, 0 XbaI, 0 XhoI restriction sites, and having a restriction map of: 
According to still another aspect of the present invention, there is provided a transformant obtainable by introducing a recombinant DNA comprising the DNA fragment having a nucleotide sequence of SEQ ID NO: 1 and ligated to a vector which can replicate or maintained in the host.
According to still another aspect of the present invention, there is provided a transformant obtainable by introducing into a host microorganism a recombinant DNA which comprises a vector, a promoter, and a DNA fragment, wherein the vector and the promoter are functionally ligated to the DNA fragment to enable expression of the toluene monooxygenase encoded by the DNA fragment in the host,
wherein the DNA fragment comprises a region sequence of SEQ ID NO: 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO: 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO: 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO: 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO: 7 of the Sequence Listing.
Further, according to still another aspect of the present invention, there is provided a transformant obtainable by introducing into a host microorganism a recombinant DNA comprising a vector, a promoter, a first DNA fragment, and a second DNA fragment;
wherein the first DNA encodes a toluene monooxygenase and comprises a region encoding a polypeptide TomL having an amino acid sequence of SEQ ID NO: 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO: 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO: 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO: 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO: 7 of the Sequence Listing,
the second DNA fragment encodes a polypeptide TomK having an amino acid sequence of SEQ ID NO: 2 and a property to enhance the toluene monooxygenase activity of a protein comprised of at least TomL to TomP; or encodes a variant TomK polypeptide in which the amino acid sequence of SEQ ID NO: 2 is altered with the proviso that the property to enhance the toluene monooxygenase activity is not impaired;
the first DNA fragment is functionally linked to the promoter to express the toluene monooxygenase activity, and the second DNA fragment is functionally linked to the promoter to express the property to enhance the activity of the toluene monooxygenase encoded by the first DNA fragment.
Further, according to still another aspect of the present invention there is provided a transformant obtainable by introducing into a host microorganism a recombinant DNA comprising a vector, a first promoter and a first DNA fragment, and a second promoter and a second DNA fragment;
wherein the first DNA encodes a toluene monooxygenase and comprises a region encoding a polypeptide TomL having an amino acid sequence of SEQ ID NO: 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO: 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO: 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO: 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO: 7 of the Sequence Listing,
the second DNA fragment encodes a polypeptide TomK having an amino acid sequence of SEQ ID NO: 2 and a property to enhance the toluene monooxygenase activity of a protein comprised of at least TomL to TomP; or encodes a variant TomK polypeptide in which the amino acid sequence of SEQ ID NO: 2 is altered with the proviso that the property to enhance the toluene monooxygenase activity is not impaired;
the first DNA fragment is functionally linked to the first promoter to express the toluene monooxygenase activity, and the second DNA fragment is functionally linked to the second promoter to express the property to enhance the activity of the toluene monooxygenase encoded by the first DNA fragment.
According to still another aspect of the present invention, there is provided a method for producing a toluene monooxygenase, which comprises a step of making an above transformant produce a toluene monooxygenase being a gene product of the DNA fragment introduced into the transformant.
According to still another aspect of the present invention, there is provided a method for degrading at least either of a chlorinated aliphatic hydrocarbon compound or an aromatic compound, which comprises a step of degrading at least either of the chlorinated aliphatic hydrocarbon compound or aromatic compound using the transformant according to any one of the aspects of the present invention mentioned above.
According to still another aspect of the present invention, there is provided a method for remedying an environment polluted with at least either of a chlorinated aliphatic hydrocarbon compound or an aromatic compound as a pollutant, comprising a step of degrading the pollutants using the transformant according to any one of the aspects of the present invention mentioned above.
According to still another aspect of the present invention, there is provided a component polypeptide having any one of amino acid sequences of SEQ ID Nos: 2-8, which can constitute a toluene monooxygenase.
According to still another aspect of the present invention, there is provided a toluene monooxygenase comprising at least component polypeptides TomL-TomP of amino acid sequences of SEQ ID NOs: 3-7.
According to still another aspect of the present invention, there is provided a variant toluene monooxygenase obtainable by mutating the above mentioned toluene monooxygenase not to loose the enzyme activity.